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The present invention relates to rapid methods for detection and quantification of microorganisms in food products for human consumption or use, and more particularly to rapid methods for the detection and quantification of sulfide-producing bacteria in fish products.
Among the most predominant bacteria associated with spoilage of fish products, and food products in general are sulfide-producing bacteria (SPB) such as Shewanella putrefaciens. SPB are especially responsible for spoilage of many kinds of foods such as seafood, including fish, fish products, mussels, mussel products, shellfish and shellfish products and other meat products such as poultry (e.g., chicken and turkey), pork, beef, and lamb, and even dairy products such as cheese. SPB are particularly responsible for spoilage in fresh or cooled aerobically-packed seafood. These bacteria are present in seawater and on the surface of all living fish and shellfish, and are transferred to the flesh during catch and processing. They grow to high levels and cause spoilage even when the fish are stored on ice (at approximately 0-4xc2x0 C.). The spoilage is mainly due to growth of psychrotropic bacteria, including S. putrefaciens. 
Traditionally, microbial analysis of fish has been limited to total viable counts. Indicator testing for total viable organisms and coliforms are the most widely used tests for routine monitoring of microbial contamination. However, during the last decade, it has been discovered that the shelf life of fish and shellfish is correlated to the level of specific spoilage bacteria and not to the count of total viable organisms. Spoilage is sensory detectable when the number of sulfide producing bacteria exceeds 107 colony forming units per gram (cfu/g) of fish muscle. In the case of cod from the North Atlantic, for example, this level is reached after approximately 12 days when the fish are stored on ice, after approximately 7 days of storage at 4xc2x0 C., and after even shorter times when stored at higher temperatures. The growth of bacteria is exponential, and the shelf life of the fish can be predicted from the number of SPB in the fish, and the growth rate of these bacteria at the actual storage temperature. In addition to sulfide production, SPB contribute to the accumulation of trimethylamine (TMA) in the fish. TMA is a primary component of unpleasant fishy odors.
Traditional agar plate methods have been developed for analysis of S. putrefaciens and other SPB in fish (Gram et al., 1987). In those methods, a non-specific growth medium is supplemented with thiosulfate, cysteine and ferricitrate. Bacteria that are able to produce sulfide from thiosulfate or cysteine appear as black colonies on the agar, due to precipitation of ferrous sulfide. The detection is therefore directly related to the spoilage property of the bacteria. This is a benefit in analysis of spoilage bacteria, since the spoilage potential of the microbe is more important that its identity. However, the agar plate method has the same disadvantages as all other agar plate methods. For instance, it does not give the possibility for early warning and it requires laboratory facilities, including an autoclave and technicians skilled in sterile technique. The method is also time and labor intensive. Also, the lowest detection level is about 100 cfu/g, which is not adequate for presence/absence tests. Furthermore, the required analysis time is at least one to three days depending on the incubation temperature. Since the analysis takes so long, this method of analysis cannot be used to make a timely decision regarding whether or not fish should be purchased, sold, or used, or whether or not the fish can be used to produce other products. Therefore, more rapid methods of detection are desired to be able to expedite the decision-making process regarding how food products such as fish and shellfish which are contaminated with sulfide-producing bacteria can be used, sold, or purchased.
Other, more rapid, analyses based on bacterial growth with a possibility for early warning have been developed for total viable counts, for several hygienic indicator bacteria and for certain pathogens. For example, a sample is inoculated in a growth medium which is more or less specific for the bacterium that is going to be detected or quantified. The medium has an indicator that is a specific substrate for the desired bacterium. The product produced by turnover of this substrate becomes detectable (for example by fluorescence) when the number of microbes reaches a certain level. Other examples are impedimentary methods, where the indication is related to a change in the number of charged molecules. In these methods, the number of microbes in the sample is calculated indirectly based on the time required until detection and on the growth rate at the given conditions. However, all of these methods suffer from one or more deficiencies, and there continues to be a need for methods which will more rapidly detect and/or quantify SPB which cause product spoilage.
The present invention comprises methods for rapidly detecting and quantifying sulfide-producing bacteria, for instance S. putrefaciens, in a sample of a food product comprising meat, dairy or fish. The food sample is combined with a quantity of growth medium. The growth medium preferably comprises an iron compound (ferric citrate or ferrous sulfate, for example) and organic and/or inorganic sulfur compounds (cysteine and sodium thiosulfate, for example) and forms an iron precipitate (e.g., iron sulfide) when exposed to S. putrefaciens or other sulfide-producing bacteria. The growth medium and the sample forms an incubation mixture. The incubation mixture is incubated for a predetermined incubation period, for example from about four or five hours to 16-18 hours.
The level of SPB in the sample is determined by using a visual detection method to identify a color change, or by using fluorescence detection methods which detect trends in fluorescence production of the incubation mixtures which are correlated with SPB numbers.